Mycophenolic acid (MPA) is a metabolite of the pro-drug mycophenolate mofetil (MMF, CellCept®), widely used for the prevention of rejection in patients receiving renal, heart, or liver transplants. Chemically, MMF is 2-morpholinoethyl(E)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate. After administration, MMF is rapidly absorbed and hydrolyzed essentially completely to MPA. Biochemically, MPA is a potent and specific inhibitor of inosine-monophosphate dehydrogenase (IMPDH), an enzyme necessary for de novo purine synthesis used by B- and T-lymphocytes. The inhibition of IMPDH by MPA at the cellular level suppresses proliferation of B- and T-lymphocytes, due to their dependency on de novo purine synthesis and, thus, results in immunosuppression at the organism level. MPA is, at times, co-administered with cyclosporine or tacrolimus in transplant patients to further enhance immunosuppression.
MPA has an acceptable therapeutic window in the range of 1–3.5 mg/L in serum or plasma. Because of its immunosuppressive effects, circulating levels of MPA beyond this range are associated with an increased risk of infections or leukopenia. Therefore, levels of MPA must be monitored to insure effective use of the drug while minimizing the risk of adverse side effects in patients.
MPA is further metabolized by UDP-glucuronosyl transferases primarily to a phenolic glucuronide, 7-O-MPAG, which is pharmacologically inactive. However, a minor metabolite, acyl glucuronide (AcMPAG), has been described to have activity similar to that of MPA with regards to inhibiting IMPDH, possibly contributing to the adverse effects of MPA therapy. See Shipkova et al., British Journal of Pharmacology 132:1027–1034 (2001). Therefore, therapeutic drug monitoring in patients receiving MPA is likely improved if levels of the active metabolite, AcMPAG, are also measured as part of the active MPA concentration, while the inactive metabolite, 7-O-MPAG, remains undetectable by the assay.
In developing an immunoassay for the detection of a small molecule target, such as a drug, which often lacks inherent antigenicity, an immunogenic compound must first be made. When the target drug is an immunosuppressant, the challenge to make an immunogenic compound increases. Typically, larger antigenic proteins, polypeptides, or other antigenic biomolecules are conjugated to the drug. Still, the immunogen must be capable of stimulating production of an antibody that interacts specifically with at least a portion of the target drug and not just the conjugated antigenic biomolecule.
Further, detection of a drug in an immunoassay generally requires the use of a detectable component or label including, but not limited to, radioisotopes, enzymes, fluorescent molecules, and particles. The label is typically conjugated to an antibody or antibody fragment, in the case of a sandwich immunoassay, or to the drug or analog of the drug, in the case of a competitive immunoassay.
The chemical structure of MPA is represented by the formula:

Modifications of MPA, for the purpose of developing immunoassays, have been previously described. U.S. Pat. No. 6,225,073 to Alexander et al., incorporated herein in entirety, teaches compounds comprising MPA bound to a polypeptide by replacement of one or more hydrogen atoms in various functional groups. For example, modifications of MPA at existing functional groups, such as the phenolic hydroxy group at position 4 or the carboxyl group at position 1′, are disclosed. Also, the methoxy group at position 6 of the isobenzofuranyl ring system may be cleaved to form a phenolic hydroxy group that is reacted to form an ether linkage. Alternatively, a functional group can be added via oxidation of a C—H bond, for example, the methyl group at position 7 of the isobenzofuranyl ring. Further, Alexander et al. used immunogens prepared by conjugating keyhole limpet hemocyanin (KLH) to MPA via substitution of the hydrogen atom in the phenolic hydroxy group at position 4, or via oxidation of the methyl group at position 7, to produce monoclonal antibodies capable of specifically binding MPA but little or no cross-reactivity to mycophenolic acid glucuronide (MPAG).
U.S. Pat. No. 6,524,808 to Dorn et al., incorporated herein in entirety, discloses conjugates made via attachment of ligands to MPA at a carbon atom in the hexanoic chain. Such conjugates were used for the purposes of making enzyme conjugates for a homogeneous enzyme inhibition immunoassay, not for immunogenic conjugates.